Methods of detecting endostatin protein

ABSTRACT

An inhibitor of endothelial cell proliferation, capable of inhibiting angiogenesis and causing tumor regression, that is approximately 20 kDa and corresponds to a C-terminal fragment of collagen type XVIII, and methods of treating angiogenesis-related disease.

CROSS REFERENCE TO PRIOR RELATED CASES

[0001] This application claims priority to provisional applicationSerial No. 60/005,835 filed Oct. 23, 1995; provisional applicationSerial No. 60/023,070 filed Aug. 2, 1996; and provisional applicationSerial No. 60/026,263 filed Sep. 17, 1996. Each of the above-referencedapplications is incorporated herein in its entirety.

TECHNICAL FIELD

[0002] This application relates to a novel inhibitor of angiogenesisuseful for treating angiogenesis-related diseases, such asangiogenesis-dependent cancer. The invention further relates to a novelcomposition and method for curing angiogenesis-dependent cancer. Inaddition, the present invention relates to diagnostic assays and kitsfor endostatin measurement, to histochemical kits for localization ofendostatin, to molecular probes to monitor endostatin biosynthesis, toantibodies that are specific for the endostatin, to the development ofpeptide agonists and antagonists to the endostatin receptor, and tocytotoxic agents linked to endostatin peptides.

BACKGROUND OF THE INVENTION

[0003] Several lines of direct evidence now suggest that angiogenesis isessential for the growth and persistence of solid tumors and theirmetastases (Folkman, 1989; Hori et al., 1991; Kim et al., 1993; Millaueret al., 1994). To stimulate angiogenesis, tumors upregulate theirproduction of a variety of angiogenic factors, including the fibroblastgrowth factors (FGF and BFGF) (Kandel et al., 1991) and vascularendothelial cell growth factor/vascular permeability factor (VEGF/VPF).However, many malignant tumors also generate inhibitors of angiogenesis,including angiostatin and thrombospondin (Chen et al., 1995; Good etal., 1990; O'Reilly et al., 1994). It is postulated that the angiogenicphenotype is the result of a net balance between these positive andnegative regulators of neovascularization (Good et al., 1990; O'Reillyet al., 1994; Parangi et al., 1996; Rastinejad et al., 1989). Severalother endogenous inhibitors of angiogenesis have been identified,although not all are associated with the presence of a tumor. Theseinclude, platelet factor 4 (Gupta et al., 1995; Maione et al., 1990),interferon-alpha, interferon-inducible protein 10 (Angiolillo et al.,1995; Strieter et al., 1995), which is induced by interleukin-12 and/orinterferon-gamma (Voest et al., 1995), gro-beta (Cao et al., 1995), andthe 16 kDa N-terminal fragment of prolactin (Clapp et al., 1993). Theonly known angiogenesis inhibitor which specifically inhibitsendothelial cell proliferation is angiostatin (O'Reilly et al. 1994).

[0004] Angiostatin is an approximately 38 kiloDalton (kDa) specificinhibitor of endothelial cell proliferation. Angiostatin is an internalfragment of plasminogen containing at least three of the five kringlesof plasminogen Angiostatin has been shown to reduce tumor weight and toinhibit metastasis in certain tumor models. (O'Reilly et al., 1994). Asit is used hereinafter, the term “angiostatin” refers to angiostatin asdescribed above; peptide fragments of angiostatin that have endothelialcell proliferation inhibiting activity; and analogs of angiostatin thathave substantial sequence homology (as defined herein) to the amino acidsequence of angiostatin, which have endothelial cell proliferationinhibiting activity.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a novel protein inhibitor, andmethod for its use. The protein is a potent and specific inhibitor ofendothelial proliferation and angiogenesis. Systemic therapy with theinhibitor causes a nearly complete suppression of tumor-inducedangiogenesis, and it exhibits strong anti-tumor activity.

[0006] The inhibitory protein has a molecular weight of approximately18,000 to approximately 20,000 Daltons (18 to 20 kDa) and is capable ofinhibiting endothelial cell proliferation in cultured endothelial cells.The protein can be further characterized by its preferred N-terminalamino acid sequence, the first twenty (20) of which are as follows: HisThr His Gln Asp Phe Gln Pro Val Leu (SEQ ID NO:1)1   2   3   4   5   6   7   8   9  10 His Leu Val Ala Leu Asn Thr ProLeu Ser 11  12  13  14  15  16  17  18  19  20

[0007] A preferred endothelial cell proliferation inhibitor of theinvention is a protein having the above-described characteristics, andwhich can be isolated and purified from the murine hemangioendotheliomacell line EOMA. This inhibitory protein has been named endostatin.

[0008] The present invention provides methods and compositions fortreating diseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal with the undesiredangiogenesis a composition comprising a substantially purifiedendostatin or endostatin derivative in a dosage sufficient to inhibitangiogenesis. The present invention is particularly useful for treatingor for repressing the growth of tumors. Administration of endostatin toa human or animal with prevascularized metastasized tumors prevents thegrowth or expansion of those tumors.

[0009] The present invention also includes diagnostic methods and kitsfor detection and measurement of endostatin in biological fluids andtissues, and for localization of endostatin in tissues. The diagnosticmethod and kit can be in any configuration well known to those ofordinary skill in the art. The present invention also includesantibodies specific for the endostatin and antibodies that inhibit thebinding of antibodies specific for the endostatin. These antibodies canbe polyclonal antibodies or monoclonal antibodies. The antibodiesspecific for endostatin can be used in diagnostic kits to detect thepresence and quantity of endostatin which is diagnostic or prognosticfor the occurrence or recurrence of cancer or other disease mediated byangiogenesis. Antibodies specific for endostatin may also beadministered to a human or animal to passively immunize the human oranimal against endostatin, thereby reducing angiogenic inhibition.

[0010] The present invention also includes diagnostic methods and kitsfor detecting the presence and quantity of antibodies that bindendostatin in body fluids. The diagnostic method and kit can be in anyconfiguration well known to those of ordinary skill in the art.

[0011] The present invention also includes endostatin peptide fragmentsthat can be labeled isotopically or with other molecules or proteins foruse in the detection and visualization of endostatin binding sites withstate of the art techniques, including, but not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

[0012] These endostatin peptides also act as agonists and antagonists atthe endostatin receptor, thereby enhancing or blocking the biologicalactivity of endostatin. Such peptides are used in the isolation of theendostatin receptor.

[0013] The present invention also includes endostatin, endostatinfragments, endostatin antisera, or endostatin receptor agonists andantagonists linked to cytotoxic agents for therapeutic and researchapplications.

[0014] The present invention includes molecular probes for theribonucleic acid and deoxyribonucleic acid involved in transcription andtranslation of endostatin. These molecular probes provide means todetect and measure endostatin biosynthesis in tissues and cells.

[0015] A surprising discovery is that various forms of recombinantendostatin protein can serve as sustained release anti-angiogenesiscompounds when administered to a tumor-bearing animal. A preferred formof the sustained release compound is un-refolded recombinantly producedendostatin.

[0016] Additionally, the present invention encompasses nucleic acidsequences comprising corresponding nucleotide codons that code for theabove disclosed amino acid sequence and for endostatin and endothelialcell proliferation inhibiting peptide fragments thereof.

[0017] The present invention also relates to methods of using theendostatin protein and peptide fragments, corresponding nucleic acidsequences, and antibodies that bind specifically to the inhibitor andits peptides, to diagnose endothelial cell-related diseases anddisorders.

[0018] The invention further encompasses a method for identifyingreceptors specific for endostatin, and the receptor molecules identifiedand isolated thereby.

[0019] The invention also relates to a method for identifying novelenzymes capable of releasing endostatin from collagen type XVIII, andother molecules containing an endostatin amino acid sequence, andpeptides thereof. Such endostatin producing enzymes are also an aspectof the invention.

[0020] An important medical method is a new form of birth control,wherein an effective amount of endostatin is administered to a femalesuch that uterine endometrial vascularization is inhibited and embryoimplantation cannot occur, or be sustained.

[0021] A particularly important aspect of the present invention is thediscovery of a novel and effective method for treatingangiogenesis-related diseases, particularly angiogenesis-dependentcancer, in patients, and for curing angiogenesis-dependent cancer inpatients. The method unexpectedly provides the medically importantresult of inhibition of tumor growth and reduction of tumor mass. Themethod relates to the co-administration of the endostatin of the presentinvention and another anti-angiogenesis compound, preferrablyangiostatin. Accordingly, the present invention also includesformulations containing endostatin, and optionally angiostatin, whichare effective for treating or curing angiogenesis-dependent cancers.

[0022] Accordingly, it is an object of the present invention to providea composition comprising an endostatin protein.

[0023] It is another object of the present invention to provide a methodof treating diseases and processes that are mediated by angiogenesis.

[0024] It is yet another object of the present invention to provide adiagnostic or prognostic method and kit for detecting the presence andamount of endostatin in a body fluid or tissue.

[0025] It is yet another object of the present invention to provide amethod and composition for treating diseases and processes that aremediated by angiogenesis including, but not limited to, hemangioma,solid tumors, leukemia, metastasis, telangiectasia psoriasisscleroderma, pyogenic granuloma, myocardial angiogenesis, plaqueneovascularization, corornay collaterals, cerebral collaterals,arteriovenous malformations, ischemic limb angiogenesis, cornealdiseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, fractures, keloids,vasculogenesis, hematopoiesis, ovulation, menstruation, andplacentation.

[0026] It is another object of the present invention to provide acomposition for treating or repressing the growth of a cancer.

[0027] It is an object of present invention to provide a method fordetecting and quantifying the presence of an antibody specific for anendostatin in a body fluid.

[0028] Still another object of the present invention is to provide acomposition consisting of antibodies to endostatin that are selectivefor specific regions of the endostatin molecule.

[0029] It is another object of the present invention to provide a methodfor the detection or prognosis of cancer.

[0030] It is another object of the present invention to provide acomposition for use in visualizing and quantitating sites of endostatinbinding in vivo and in vitro.

[0031] It is yet another object of the present invention to provide acomposition for use in detection and quantification of endostatinbiosynthesis.

[0032] It is yet another object of the present invention to provide atherapy for cancer that has minimal side effects.

[0033] Still another object of the present invention is to provide acomposition comprising endostatin or an endostatin peptide linked to acytotoxic agent for treating or repressing the growth of a cancer.

[0034] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1: Inhibition of Capillary Endothelial Cell Proliferation byConditioned Media from EOMA Cells.

[0036] Conditioned media collected from confluent EOMA cells or basemedia was applied to bovine capillary endothelial cells with 1 ng/mlbFGF in a 72 hour proliferation assay. Endothelial cell proliferationwas inhibited by the EOMA conditioned media. Each bar represents themean±SEM.

[0037]FIG. 2: Purification of an Inhibitor of Endothelial Proliferationfrom EOMA Conditioned Media.

[0038] Conditioned media collected from confluent EOMA cells wasfractionated on a heparin sepharose column. Endothelial proliferationinhibiting activity eluted at approximately 0.8M NaCl.

[0039]FIG. 3: Purification of an Inhibitor of Endothelial Proliferationby Gel Filtration.

[0040] Purified inhibitor from heparin sepharose column chromatographywas applied to a gel filtration column and eluted as a single peak.

[0041]FIG. 4: Purification of Inhibitor of Endothelial CellProliferation by Reversed Phase Column Chromatography.

[0042] Inhibitor purified by heparin sepharose and gel filtrationchromatography was applied to a reverse phase column. The inhibitoreluted as a single band from the column at approximately 45% of theacetonitrile.

[0043]FIG. 5: N-terminal Amino Acid Sequence of An Inhibitor ofEndothelial Cell Proliferation.

[0044] The N-terminal sequence of the purified inhibitor of endothelialcell proliferation is shown in relation to a schematic diagram ofcollagen type 18. The N-terminal sequence revealed identity of theinhibitor to an approximately 20 kDa C-terminal fragment (shown in solidshading) for collagen type XVIII. The open boxes represent thecollagenase domains of collagen type XVIII.

[0045]FIG. 6: Treatment of Lewis Lung Carcinoma With Recombinant MouseEndostatin Inhibitor.

[0046] Recombinant inhibitor produced in E. coli was administered tomice seeded with Lewis lung carcinoma that had achieved a tumor volumeof approximately 150 mm³. The inhibitor was administered at 20mg/kg/day. Tumor mass regressed to non-detectable levels afterapproximately 12 days of treatment.

[0047]FIG. 7: Systemic Therapy with Recombinant Endostatin RegressesLewis Lung Carcinoma Primary Tumors.

[0048] (A) Mice were implanted subcutaneously on the dorsum with Lewislung carcinoma cells. Systemic therapy with recombinant mouse endostatin(20 mg/kg/day) was begun when tumors were approximately 200 mm³ (1% ofbody weight). Tumors in the mice treated with endostatin inhibitorrapidly regressed and were inhibited by >99% relative to saline-treatedcontrols. Each point represents mean±SEM for 5 mice. The experiment wasrepeated with comparable results.

[0049] (B) Representative treated and untreated tumor-bearing mice after11 days of systemic therapy with endostatin. Saline-treated mice (right)had rapidly growing red tumors with ulcerated surfaces. Endostatintreated mice (left) had small pale residual tumors (arrow).

[0050] (C) Residual disease in endostatin treated mice. Three of thefive endostatin treated mice were sacrificed after 16 days of therapy.Autopsy revealed small white residual tumors at the site of the originalprimary implantation (arrows).

[0051]FIG. 8: Treatment of Murine T241 Fibrosarcoma with RecombinantMouse Endostatin from E. coli.

[0052] Mice were seated with T241 Fibrosarcoma cells. Control mice weretreated with saline. Experimental mice were treated with 20 mg/kg/day ofrecombinant mouse Endostatin directed from E. coli.

[0053]FIG. 9: Treatment of Murine B16F10 Melanoma with Recombinant MouseEndostatin from E. coli.

[0054] Mice were seated with Murine B16F10 melanoma cells. Controlanimals were treated with saline. Experimental animals were treated with20 mg/kg/day of recombinant mouse Endostatin direct from E. coli.

[0055]FIG. 10: Treatment of EOMA Hemangioendothelioma with RecombinantMouse Endostatin from E. coli.

[0056] Mice were seated with EOMA hemangioendothelioma cells. Controlanimals were treated with saline. Experimental animals were treated with20 mg/kg/day of Recombinant Mouse Endostatin direct from E. coli.

[0057]FIG. 11: Treatment of Lewis Lung Carcinoma with Recombinant Mouseor Human Endostatin direct from E. coli.

[0058] Mice were seated with Lewis Lung Carcinoma cells. Control animalswere treated with saline. Experimental animals were treated withRecombinant Endostatin derived from the mouse sequence or RecombinantEndostatin direct from the human sequence, wherein both Endostatin areproduced recombinantly in the E. coli. Mouse Endostatin was administeredat either 20 mg/kg/day or 2.5 mg/kg/day, and Human Endostatin wasadministered at 20 mg/kg/day.

[0059]FIG. 12: Endostatin Results in an Inhibition of Angiogenesis andan Increase in Apoptosis of Lewis Lung Carcinoma Primary Tumors.

[0060] Histological sections of tumors from saline versus endostatintreated mice implanted with Lewis lung carcinomas were analyzed forproliferation (PCNA), apoptosis (TUNEL), and angiogenesis (vWF). Therewas no significant difference in the proliferative index of tumor cells(A) in treated versus untreated tumors. In contrast, the apoptotic indexof the tumor cells (B) increased 8-fold (p<0.001) in the endostatintreated mice. Vessel density (C) was determined by counting the numberof capillary blood vessels per high-power field (HPF) in sectionsstained with antibodies against vWF. Angiogenesis was almost completelysuppressed in the residual microscopic tumors of the endostatin treatedmice (p<0.001).

[0061]FIG. 13: Cycle Dormancy Therapy of Lewis Lung Carcinoma withRecombinant Mouse Endostatin From E. Coli.

[0062] Mice were implanted subcutaneously on the dorsum with Lewis lungcarcinoma cells. Systemic therapy with recombinant mouse inhibitor(endostatin), administered at a dose of 20 mg/kg/day, was begun whentumors were approximately 200 mm³ (1% of body weight). Tumors in themice treated with the endostatin inhibitor rapidly regressed toessentially non-detectable levels after approximately 15 days oftherapy. When treatment was terminated the tumor volume increasedrapidly and was subsequently treatable to the same non-detectable levelsby reinitiation of treatment. The peaks and valleys in the figure showthe cycling effect of inhibition with endostatin.

[0063]FIG. 14: Combination Therapy with Recombinant Mouse Angiostatinand Endostatin from E. Coli.

[0064] Mice were implanted subcutaneously on the dorsum with Lewis lungcarcinoma cells. Systemic therapy with a combination of recombinantmouse endostatin (20 mg/kg/day) and recombinant mouse angiostatin (20mg/kg/day) was begun when tumors were approximately 300 mm³. Tumors inthe mice treated with the combination therapy rapidly regressed toessentially non-detectable level in about 15 days. Importantly, theregressed tumors remained dormant and did not increase in size or massafter treatment was stopped. This is an unexpected result of substatnialmedical significance.

DETAILED DESCRIPTION OF THE INVENTION

[0065] Applicants have discovered a new class of protein molecules thathave the ability to inhibit endothelial proliferation when added toproliferating endothelial cells in vitro. Accordingly, these proteinmolecules have been functionally defined as endostatins, however, it isto be understood that this functional definition is no way limits thebioactivity of endostatins to inhibition of endothelial cell growth invitro or in vivo. Many other functions of endostatins are likely.

[0066] The term “endostatin” refers to a protein that is preferably 18kDa to 20 kDa in size as determined by non-reduced and reduced gelelectrophoresis, respectively. The term endostatin also includesprecursor forms of the 18 kDa to 20 kDa protein. The term endostatinalso includes fragments of the 18 kDa to 20 kDa protein and modifiedproteins and peptides that have a substantially similar amino acidsequence, and which are capable inhibiting proliferation of endothelialcells. For example, silent substitutions of amino acids, wherein thereplacement of an amino acid with a structurally or chemically similaramino acid does not significantly alter the structure, conformation oractivity of the protein, is well known in the art. Such silentsubstitutions are intended to fall within the scope of the appendedclaims.

[0067] It will be appreciated that the term “endostatin” includesshortened proteins or peptides wherein one or more amino acid is removedfrom either or both ends of endostatin, or from an internal region ofthe protein, yet the resulting molecule retains endothelialproliferation inhibiting activity. The term “endostatin” also includeslengthened proteins or peptides wherein one or more amino acid is addedto either or both ends of endostatin, or to an internal location in theprotein, yet the resulting molecule retains endothelial proliferationinhibiting activity. Such molecules, for example with tyrosine added inthe first position are useful for labelling such as radioiodination with¹²⁵iodine for use in assays. Labelling with other radioisotopes may beuseful in providing a molecular tool for destroying the target cellcontaining endostatin receptors. Other labelling with molecules such asricin may provide a mechanism for destroying cells with endostatinreceptors.

[0068] “Substantial sequence homology” means at least approximately 70%homology between amino acid residue seqence in the endostatin analogsequence and that of endostatin, preferably at least approximately 80%homology, more preferably at least approximately 90% homology.

[0069] Also included in the definition of the term endostatin aremodifications of the endostatin protein, its subunits and peptidefragments. Such modifications include substitutions of naturallyoccurring amino acids at specific sites with other molecules, includingbut not limited to naturally and non-naturally occurring amino acids.Such substitutions may modify the bioactivity of endostatin and producebiological or pharmacological agonists or antagonists. The termendostatin also includes an N terminal fragment of endostatin consistingof the sequence of the first 20 N terminal amino acids which is shown inSEQ ID NO:1 and is shown in Table 1. This sequence of the first 20 Nterminal amino acids corresponds to a C-terminal fragment of newlyidentified collagen type XVIII.

[0070] Table 1 shows the correspondence of 3 letter and 1 letter aminoacid designations. TABLE 1 Amino Acid Residue Abbreviation 1 HIS H 2 THRT 3 HIS H 4 GLN Q 5 ASP D 6 PHE F 7 GLN Q 8 PRO P 9 VAL V 10 LEU L 11HIS H 12 LEU L 13 VAL V 14 ALA A 15 LEU L 16 ASN N 17 THR T 18 PRO P 19LEU L 20 SER S

[0071] The N-terminal amino acid sequence of endostatin coresponds to aninternal 20 amino acid peptide fragment found in mouse collagen alpha 1type XVIII starting at amino acid 1105 and ending at amino acid 1124.The N-terminal amino acid sequence of the inhibitor also corresponds toan internal 20 amino acid peptide fragment found in human collagen alpha1 type XVIII starting at amino acid 1132 and ending at amino acid 1151.

[0072] Endostatin can be isolated from murine hemangioendothelioma EOMA.Endostatin may be produced from recombinant sources, from geneticallyaltered cells implanted into animals, from tumors, and from cellcultures as well as other sources. It is anticipated that endostatin ismade in cells of the nervous system. Endostatin can be isolated frombody fluids including, but not limited to, serum, urine and ascites, orsynthesized by chemical or biological methods (e.g. cell culture,recombinant gene expression, peptide synthesis,—and in vitro enzymaticcatalysis of precursor molecules to yield active endostatin).Recombinant techniques include gene amplification from DNA sources usingthe polymerase chain reaction (PCR), and gene amplification from RNAsources using reverse transcriptase/PCR.

[0073] Endostatin specifically and reversibly inhibits endothelial cellproliferation. The inhibitor protein molecules of the invention areuseful as a birth control drug, and for treating angiogenesis-relateddiseases, particularly angiogenesis-dependent cancers and tumors. Theprotein molecules are also useful for curing angiogenesis-dependentcancers and tumors. The unexpected and surprising ability of these novelcompounds to treat and cure angiogenesis-dependent cancers and tumorsanswers a long felt unfulfilled need in the medical arts, and providesan important benefit to mankind.

[0074] Important terms that are used herein are defined as follows.“Cancer” means angiogenesis-dependent cancers and tumors, i.e. tumorsthat require for their growth (expansion in volume and/or mass) anincrease in the number and density of the blood vessels supplying thenwith blood. “Regression” refers to the reduction of tumor mass and size.

[0075] The endothelial proliferation inhibiting proteins of the presentinvention can be made by automated protein synthesis methodologies wellknown to one skilled in the art. Alternatively, endothelialproliferation inhibiting proteins, or endostatins, of the presentinvention may be isolated from larger known proteins, such as humanalpha 1 type XVIII collagen and mouse alpha 1 type XVIII collagen,proteins that share a common or similar N-terminal amino acid sequence.Examples of other potential endostatin source materials having similarN-terminal amino acid sequences include Bos taurus pregastric esterase,human alpha 1 type 15 collagen, NAD-dependent formate dehydrogenase (EC1.2.1.2) derived from Pseudomonas sp., s11459 hexon protein of bovineadenovirus type 3, CELF21D12 2 F21d12.3 Caenorhabditis elegans geneproduct, VAL1 TGMV AL1 protein derived from tomato golden mosaic virus,s01730 hexon protein derived from human adenovirus 12, Saccharomycescerevisiae. For example, peptides closely related to endostatin may bederived from BOVMPE 1 pregastric esterase (BOS TAURUS) gene sequencecorresponding to amino acids 502 to 521, and collagen alpha 1 type 15from humans beginning at amino acid 316 ending at 335.

[0076] Proteins and peptides derived from these and other sources,including manual or automated protein synthesis, may be quickly andeasily tested for endothelial proliferation inhibiting activity using abiological activity assay such as the bovine capillary endothelial cellproliferation assay. Other bioassays for inhibiting activity include thechick CAM assay, the mouse corneal assay, and the effect ofadministering isolated or synthesized proteins on implanted tumors. Thechick CAM assay is described by O'Reilly, et al. in “AngiogenicRegulation of Metastatic Growth” Cell, vol. 79 (2), Oct. 21, 1994, pp.315-328, which is hereby incorporated by reference in its entirety.Briefly, 3 day old chicken embryos with intact yolks are separated fromthe egg and placed in a petri dish. After 3 days of incubation amethylcellulose disc containing the protein to be tested is applied tothe CAM of individual embryos. After 48 hours of incubation, the embryosand CAMs are observed to determine whether endothelial growth has beeninhibited. The mouse corneal assay involves implanting a growthfactor-containing pellet, along with another pellet containing thesuspected endothelial growth inhibitor, in the cornea of a mouse andobserving the pattern of capillaries that are elaborated in the cornea.

[0077] Applicants' invention also encompasses nucleic acid sequencesthat correspond to and code for the endothelial proliferation-inhibitingprotein molecules of the invention, and to monoclonal and polyclonalantibodies that bind specifically to such protein molecules. Thebiologically active protein molecules, nucleic acid sequencescorresponding to the proteins, and antibodies that bind specifically tothe proteins of the present invention are useful for modulatingendothelial processes in vivo, and for diagnosing and treatingendothelial cell-related diseases, for example by gene therapy.

[0078] Nucleic acid sequences that correspond to, and code for,endostatin and endostatin analogs can be prepared based upon theknowledge of the amino acid sequence, and the art recognizedcorrespondence between codons (sequences of three nucleic acid bases),and amino acids. Because of the degeneracy of the genetic code, whereinthe third base in a codon may vary yet still code for the same aminoacid, many different possible coding nucleic acid sequences arederivable for any particular protein or peptide fragment.

[0079] Nucleic acid sequences are synthesized using automated systemswell known in the art. Either the entire sequence may be synthesized ora series of smaller oligonucleotides are made and subsequently ligatedtogether to yield the full length sequence. Alternatively, the nucleicacid sequence may be derived from a gene bank using oligonucleotidesprobes designed based on the N-terminal amino acid sequence and wellknown techniques for cloning genetic material.

[0080] The present invention also includes the detection of endostatinin body fluids and tissues for the purpose of diagnosis or prognosis ofangiogenesis-mediated diseases such as cancer. The present inventionalso includes the detection of endostatin binding sites and receptors incells and tissues. The present invention also includes methods oftreating or preventing angiogenic diseases and processes including, butnot limited to, arthritis and tumors by stimulating the production ofendostatin, and/or by administering substantially purified endostatin,or endostatin agonists or antagonists, and/or endostatin antisera orantisera directed against endostatin antisera to a patient. Additionaltreatment methods include administration of endostatin, endostatinfragments, endostatin antisera, or endostatin receptor agonists andantagonists linked to cytotoxic agents. It is to be understood that theendostatin can be animal or human in origin. Endostatin can also beproduced synthetically by chemical reaction or by recombinant techniquesin conjunction with expression systems. Endostatin can also be producedby enzymatically cleaving different molecules, including endostatinprecursors, containing sequence homology or identity with segments ofendostatin to generate peptides having anti-angiogenesis activity.

[0081] Passive antibody therapy using antibodies that specifically bindendostatin can be employed to modulate endothelial-dependent processessuch as reproduction, development, and wound healing and tissue repair.In addition, antisera directed to the Fab regions of endostatinantibodies can be administered to block the ability of endogenousendostatin antisera to bind endostatin.

[0082] Antibodies specific for endostatin and endostatin analogs aremade according to techniques and protocols well known in the art. Theantibodies may be either polyclonal or monoclonal. The antibodies areutilized in well know immunoassay formats, such as competitive andnon-competitive immunoassays, including ELISA, sandwich immunoassays andradioimmunoassays (RIAs), to determine the presence or absence of theendothelial proliferation inhibitors of the present invention in bodyfluids. Examples of body fluids include but are not limited to blood,serum, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterinefluid, saliva, and mucus.

[0083] The proteins, nucleic acid sequences and antibodies of thepresent invention are useful for diagnosing and treating endothelialcell-related diseases and disorders. A particularly importantendothelial cell process is angiogenesis, the formation of bloodvessels. Angiogenesis-related diseases may be diagnosed and treatedusing the endothelial cell proliferation inhibiting proteins of thepresent invention. Angiogenesis-related diseases include, but are notlimited to, angiogenesis-dependent cancer, including, for example, solidtumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. The endothelial cell proliferation inhibitingproteins of the present invention are useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions,atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.They are also useful in the treatment of diseases that have angiogenesisas a pathologic consequence such as cat scratch disease (Rochele minaliaquintosa) and ulcers (Helobacter pylori).

[0084] The endothelial cell proliferation inhibiting proteins can beused as a birth control agent by reducing or preventing uterinevascularization required for embryo implantation. Thus, the presentinvention provides an effective birth control method when an amount ofthe inhibitory protein sufficient to prevent embryo implantation isadministered to a female. In one aspect of the birth control method, anamount of the inhibiting protein sufficient to block embryo implantationis administered before or after intercourse and fertilization haveoccured, thus providing an effective method of birth control, possible a“morning after” method. While not wanting to be bound by this statement,it is believed that inhibition of vascularization of the uterineendometrium interferes with implantation of the blastocyst. Similarinhibition of vascularization of the mucosa of the uterine tubeinterferes with implantation of the blastocyst, preventing occurrence ofa tubal pregnancy. Administration methods may include, but are notlimited to, pills, injections (intravenous, subcutaneous,intramuscular), suppositories, vaginal sponges, vaginal tampons, andintrauterine devices. It is also believed that endostatin administrationwill interfere with normal enhanced vascularization of the placenta, andalso with the development of vessels within a successfully implantedblastocyst and developing embryo and fetus.

[0085] Conversely, blockade of endostatin receptors with endostatinanalogs which act as receptor antagonists may promote endothelializationand vascularization. Such effects may be desirable in situations ofinadequate vascularization of the uterine endometrium and associatedinfertilty, wound repair, healing of cuts and incisions, treatment ofvascular problems in diabetics, especially retinal and peripheralvessels, promotion of vascularization in transplanted tissue includingmuscle and skin, promotion of vascularization of cardiac muscleespecially following transplantation of a heart or heart tissue andafter bypass surgery, promotion of vascularization of solid andrelatively avascular tumors for enhanced cytotoxin delivery, andenhancement of blood flow to the nervous system, including but notlimited to the cerebral cortex and spinal cord.

[0086] A surprising discovery is that un-refolded and non-solublerecombinant endostatin is also a potent anti-angiogenesis compound whichserves as a sustained release depot when administered to a patient.

[0087] The present invention also relates to methods of using endostatinand endothelial cell proliferation inhibiting peptide fragments ofendostatin, nucleic acid sequences corresponding to endostatin andactive peptide fragments thereof, and antibodies that bind specificallyto endostatin and its peptides, to diagnose endothelial cell-relateddiseases and disorders.

[0088] The invention further encompasses a method for identifyingendostatin-specific receptors, and the receptor molecules identified andisolated thereby.

[0089] The present invention also provides a method for quantitation ofendostatin receptors.

[0090] A particularly important aspect of the present invention is thediscovery of a novel and effective method for treating and curingangiogenesis-dependent cancer in patients. It was unexpectedly foundthat the co-administration of endostatin and angiostatin in an amountsufficient to inhibit tumor growth and cause sustainable regression oftumor mass to microscopic size cures angiogenesis-dependent cancer.Accordingly, the present invention also includes formulations effectivefor treating or curing angiogenesis-dependent cancers and tumors.

[0091] More particularly, recombinant mouse endostatin, from insectcells or E. coli, potently inhibits angiogenesis and the growth ofmetastases and primary tumors. In a novel method of sustained release,the E. coli-derived recombinant endostatin was administered as anun-refolded suspension in an amount sufficient to inhibit angiogenesis,thereby inhibiting tumor growth. Tumor mass was reduced when recombinantendostatin was administered in an amount sufficient to cause regressionof the tumor. Primary tumors of 1-2% of body weight regressed by greaterthan 150-fold to become microscopic dormant lesions when treated byendostatin. Immunohistochemical analysis of the dormant tumors revealedblocked angiogenesis accompanied by high proliferation of the tumorcells balanced by a high rate of tumor cell apoptosis. There was noevidence of toxicity in any of the mice treated with endostatin.

[0092] It is contemplated as part of the present invention thatendostatin can be isolated from a body fluid such as blood or urine ofpatients or the endostatin can be produced by recombinant DNA methods orsynthetic peptide chemical methods that are well known to those ofordinary skill in the art. Protein purification methods are well knownin the art and a specific example of a method for purifying endostatin,and assaying for inhibitor activity is provided in the examples below.Isolation of human endogenous endostatin is accomplished using similartechniques.

[0093] One example of a method of producing endostatin using recombinantDNA techniques entails the steps of (1) identifying and purifying anendostatin as discussed above, and as more fully described below, (2)determining the N-terminal amino acid sequence of the purifiedinhibitor, (3) synthetically generating a DNA oligonucleotide probe thatcorresponds to the N-terminal amino acid sequence, (4) generating a DNAgene bank from human or other mammalian DNA, (5) probing the gene bankwith the DNA oligonucleotide probe, (6) selecting clones that hybridizeto the oligonucleotide, (7) isolating the inhibitor gene from the clone,(8) inserting the gene into an appropriate vector such as an expressionvector, (9) inserting the gene-containing vector into a microorganism orother expression system capable of expressing the inhibitor gene, and(10) isolating the recombinantly produced inhibitor. The abovetechniques are more fully described in laboratory manuals such as“Molecular Cloning: A Laboratory Manual” Second Edition by Sambrook etal., Cold Spring Harbor Press, 1989.

[0094] The gene for endostatin may also be isolated from cells or tissue(such as tumor cells) that express high levels of endostatin by (1)isolating messenger RNA from the tissue, (2) using reverse transcriptaseto generate the corresponding DNA sequence and then (3) using PCR withthe appropriate primers to amplify the DNA sequence coding for theactive endostatin amino acid sequence.

[0095] Yet another method of producing endostatin, or biologicallyactive fragments thereof, is by peptide synthesis. Once a biologicallyactive fragment of an endostatin is found using the assay systemdescribed more fully below, it can be sequenced, for example byautomated peptide sequencing methods. Alternatively, once the gene orDNA sequence which codes for endostatin is isolated, for example by themethods described above, the DNA sequence can be determined, which inturn provides information regarding the amino acid sequence. Thus, ifthe biologically active fragment is generated by specific methods, suchas tryptic digests, or if the fragment is N-terminal sequenced, theremaining amino acid sequence can be determined from the correspondingDNA sequence.

[0096] Once the amino acid sequence of the peptide is known, for examplethe N-terminal 20 amino acids, the fragment can be synthesized bytechniques well known in the art, as exemplified by “Solid Phase PeptideSynthesis: A Practical Approach” E. Atherton and R. C. Sheppard, IRLPress, Oxford England. Similarly, multiple fragments can be synthesizedwhich are subsequently linked together to form larger fragments. Thesesynthetic peptide fragments can also be made with amino acidsubstitutions at specific locations in order to test for agonistic andantagonistic activity in vitro and in vivo. Peptide fragments thatpossess high affinity binding to tissues can be used to isolate theendostatin receptor on affinity columns. Isolation and purification ofthe endostatin receptor is a fundamental step towards elucidating themechanism of action of endostatin. This facilitates development of drugsto modulate the activity of the endostatin receptor, the final pathwayto biological activity. Isolation of the receptor enables theconstruction of nucleotide probes to monitor the location and synthesisof the receptor, using in situ and solution hybridization technology.

[0097] Endostatin is effective in treating diseases or processes thatare mediated by, or involve, angiogenesis. The present inventionincludes the method of treating an angiogenesis mediated disease with aneffective amount of endostatin or endostatin agonists and antagonists.The angiogenesis mediated diseases include, but are not limited to,solid tumors; blood born tumors such as leukemias; tumor metastasis;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic, diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Endostatin is useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions,atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.Endostatin can be used as a birth control agent by preventingvascularization required for blastocyst implantation and for developmentof the placenta, the blastcyst, the embryo and the fetus.

[0098] The synthetic peptide fragments of endostatin have a variety ofuses. The peptide that binds to the endostatin receptor with highspecificity and avidity is radiolabeled and employed for visualizationand quantitation of binding sites using autoradiographic and membranebinding techniques. This application provides important diagnostic andresearch tools. Knowledge of the binding properties of the endostatinreceptor facilitates investigation of the transduction mechanisms linkedto the receptor.

[0099] In addition, labeling these peptides with short lived isotopesenables visualization of receptor binding sites in vivo using positronemission tomography or other modern radiographic techniques in order tolocate tumors with endostatin binding sites.

[0100] Systematic substitution of amino acids within these synthesizedpeptides yields high affinity peptide agonists and antagonists to theendostatin receptor that enhance or diminish endostatin binding to itsreceptor. Such agonists are used to suppress the growth ofmicrometastases, thereby limiting the spread of cancer. Antagonists toendostatin are applied in situations of inadequate vascularization, toblock the inhibitory effects of angiostatin and possibly promoteangiogenesis. This treatment may have therapeutic effects to promotewound healing in diabetics.

[0101] Endostatin peptides are employed to develop affinity columns forisolation of the endostatin receptor from cultured tumor cells.Isolation and purification of the endostatin receptor is followed byamino acid sequencing. Next, nucleotide probes are developed forinsertion into vectors for expression of the receptor. These techniquesare well known to those skilled in the art. Transfection of theendostatin receptor into tumor cells enhances the responsiveness ofthese cells to endogenous or exogenous endostatin and thereby decreasingthe rate of metastatic growth.

[0102] Cytotoxic agents, such as ricin, are linked to endostatin, andhigh affinity endostatin peptide fragments, thereby providing a tool fordestruction of cells that bind endostatin. These cells may be found inmany locations, including but not limited to, micrometastases andprimary tumors. Peptides linked to cytotoxic agents are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity endostatin fragments are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofendostatin antagonists may be co-applied with stimulators ofangiogenesis to increase vascularization of tissue. This therapeuticregimen provides an effective means of destroying metastatic cancer.

[0103] According to the present invention, endostatin may be used incombination with other compositions and procedures for the treatment ofdiseases. For example, a tumor may be treated conventionally withsurgery, radiation or chemotherapy combined with endostatin and thenendostatin may be subsequently administered to the patient to extend thedormancy of micrometastases and to stabilize any residual primary tumor.

[0104] The endostatin of the present invention also can be used togenerate antibodies that are specific for the inhibitor. The antibodiescan be either polyclonal antibodies or monoclonal antibodies. Theseantibodies that specifically bind to the endostatin can be used indiagnostic methods and kits that are well known to those of ordinaryskill in the art to detect or quantify the endostatin in a body fluid ortissue. Results from these tests can be used to diagnose or predict theoccurrence or recurrence of a cancer and other angiogenesis mediateddiseases.

[0105] The endostatin also can be used in a diagnostic method and kit todetect and quantify antibodies capable of binding endostatin. These kitswould permit detection of circulating endostatin antibodies whichindicates the spread of micrometastases in the presence of endostatinsecreted by primary tumors in situ. Patients that have such circulatinganti-endostatin antibodies may be more likely to develop tumors andcancers, and may be more likely to have recurrences of cancer aftertreatments or periods of remission. The Fab fragments of theseanti-endostatin antibodies may be used as antigens to generateanti-endostatin Fab-fragment antisera which can be used to neutralizethe removal of circulating endostatin by anti-endostatin antibodies.

[0106] Another aspect of the present invention is a method of blockingthe action of excess endogenous endostatin. This can be done bypassively immunizing a human or animal with antibodies specific for theundesired endostatin in the system. This treatment can be important intreating abnormal ovulation, menstruation and placentation, andvasculogenesis. This provides a useful tool to examine the effects ofendostatin removal on metastatic processes. The Fab fragment ofendostatin antibodies contains the binding site for endostatin. Thisfragment is isolated from endostatin antibodies using techniques knownto those skilled in the art. The Fab fragments of endostatin antiseraare used as antigens to generate production of anti-Fab fragment serum.Infusion of this antiserum against the Fab fragments of endostatinprevents endostatin from binding to endostatin antibodies. Therapeuticbenefit is obtained by neutralizing endogenous anti-endostatinantibodies by blocking the binding of endostatin to the Fab fragments ofanti-endostatin. The net effect of this treatment is to facilitate theability of endogenous circulating endostatin to reach target cells,thereby decreasing the spread of metastases.

[0107] It is to be understood that the present invention is contemplatedto include any derivatives of the endostatin that have endothelialinhibitory activity. The present invention includes the entireendostatin protein, derivatives of the endostatin protein andbiologically-active fragments of the endostatin protein. These includeproteins with endostatin activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups.The present invention also includes genes that code for endostatin andthe endostatin receptor, and to proteins that are expressed by thosegenes.

[0108] The proteins and protein fragments with the endostatin activitydescribed above can be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition, the endostatin may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the endostatin isslowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of endostatin throughcannulae to the site of interest, such as directly into a metastaticgrowth or into the vascular supply to that tumor. The biodegradablepolymers and their use are described, for example, in detail in Brem etal., J. Neurosurg. 74:441-446 (1991), which is hereby incorporated byreference in its entirety.

[0109] The dosage of the endostatin of the present invention will dependon the disease state or condition being treated and other clinicalfactors such as weight and condition of the human or animal and theroute of administration of the compound. For treating humans or animals,between approximately 0.5 mg/kilogram to 500 mg/kilogram of theendostatin can be administered. A more preferable range is 1 mg/kilogramto 100 mg/kilogram with the most preferable range being from 2mg/kilogram to 50 mg/kilogram. Depending upon the half-life of theendostatin in the particular animal or human, the endostatin can beadministered between several times per day to once a week. It is to beunderstood that the present invention has application for both human andveterinary use. The methods of the present invention contemplate singleas well as multiple administrations, given either simultaneously or overan extended period of time.

[0110] The endostatin formulations include those suitable for oral,rectal, ophthalmic (including intravitreal or intracameral), nasal,topical (including buccal and sublingual), intrauterine, vaginal orparenteral (including subcutaneous, intraperitoneal, intramuscular,intravenous, intradermal, intracranial, intratracheal, and epidural)administration. The endostatin formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and the pharmaceutical carrier(s)or excipient(s). In general, the formulations are prepared by uniformlyand intimately bringing into association the active ingredient withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0111] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0112] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question.

[0113] Different peptide fragments of the intact endostatin molecule canbe synthesized for use in several applications including, but notlimited to the following; as antigens for the development of specificantisera, as agonists and antagonists active at endostatin bindingsites, as peptides to be linked to cytotoxic agents for targeted killingof cells that bind endostatin. The amino acid sequences that comprisethese peptides are selected on the basis of their position on theexterior regions of the molecule and are accessible for binding toantisera. The amino and carboxyl termini of endostatin, as well as themid-region of the molecule are represented separately among thefragments to be synthesized. The amino terminus distal to the 20th aminoacid and carboxyl termini of endostatin may contain or be modified tocontain tyrosine and lysine residues and are labeled with manytechniques. A tyrosine or lysine is added to fragments that do not havethese residues to facilitate labeling of reactive amino and hydroxylgroups on the peptide. These peptide sequences are compared to knownsequences using sequence data banks to determine potential sequencehomologies. This information facilitates elimination of sequences thatexhibit a high degree of sequence homology to other molecules, therebyenhancing the potential for high specificity in the development ofantisera, agonists and antagonists to endostatin.

[0114] Peptides can be synthesized in a standard microchemical facilityand purity checked with HPLC and mass spectrophotometry. Methods ofpeptide synthesis, HPLC purification and mass spectrophotometry arecommonly known to those skilled in these arts.

[0115] Peptides and endostatin are also produced in recombinant E. coli,as described below, or in insect or yeast expression systems, andpurified with column chromatography.

[0116] Endostatin and endostatin derived peptides can be coupled toother molecules using standard methods. The amino terminus distal to the20th amino acid and the carboxyl terminus of endostatin may both containtyrosine and lysine residues and are isotopically and nonisotopicallylabeled with many techniques, for example radiolabeling usingconventional techniques (tyrosine residues—chloramine T, iodogen,lactoperoxidase; lysine residues—Bolton-Hunter reagent). These couplingtechniques are well known to those skilled in the art. The couplingtechnique is chosen on the basis of the functional groups available onthe amino acids including, but not limited to amino, sulfhydral,carboxyl, amide, phenol, and imidazole. Various reagents used to effectthese couplings include among others, glutaraldehyde, diazotizedbenzidine, carbodiimide, and p-benzoquinone.

[0117] Endostatin peptides are chemically coupled to isotopes, enzymes,carrier proteins, cytotoxic agents, fluorescent molecules and othercompounds for a variety of applications. The efficiency of the couplingreaction is determined using different techniques appropriate for thespecific reaction. For example, radiolabeling of an endostatin peptideor protein with ¹²⁵I is accomplished using chloramine T and Na¹²⁵I ofhigh specific activity. The reaction is terminated with sodiummetabisulfite and the mixture is desalted on disposable columns. Thelabeled peptide is eluted from the column and fractions are collected.Aliquots are removed from each fraction and radioactivity measured in agamma counter. In this manner, the unreacted Na¹²⁵I is separated fromthe labeled endostatin peptide. The peptide fractions with the highestspecific radioactivity are stored for subsequent use such as analysis ofthe ability to bind to endostatin antisera.

[0118] Another application of peptide conjugation is for production ofpolyclonal antisera. For example, endostatin peptides containing lysineresidues are linked to purified bovine serum albumin usingglutaraldehyde. The efficiency of the reaction is determined bymeasuring the incorporation of radiolabeled peptide. Unreactedglutaraldehyde and peptide are separated by dialysis. The conjugate isstored for subsequent use.

[0119] Antiserum against endostatin can be generated. After peptidesynthesis and purification, both monoclonal and polyclonal antisera areraised using established techniques known to those skilled in the art.For example, polyclonal antisera may be raised in rabbits, sheep, goatsor other animals. Endostatin peptides conjugated to a carrier moleculesuch as bovine serum albumin, or endostatin itself, is combined with anadjuvant mixture, emulsified and injected subcutaneously at multiplesites on the back, neck, flanks, and sometimes in the footpads. Boosterinjections are made at regular intervals, such as every 2 to 4 weeks.Blood samples are obtained by venipuncture, for example using themarginal ear veins after dilation, approximately 7 to 10 days after eachinjection. The blood samples are allowed to clot overnight at 4° C. andare centrifuged at approximately 2400×g at 4 C.° for about 30 minutes.The serum is removed, aliquoted, and stored at 4 C.° for immediate useor at −20 to −90 C.° for subsequent analysis.

[0120] All serum samples from generation of polyclonal antisera or mediasamples from production of monoclonal antisera are analyzed fordetermination of titer. Titer is established through several means, forexample, using dot blots and density analysis, and also withprecipitation of radiolabeled peptide-antibody complexes using proteinA, secondary antisera, cold ethanol or charcoal-dextran followed byactivity measurement with a gamma counter. The highest titer antiseraare also purified on affinity columns which are commercially available.Endostatin peptides are coupled to the gel in the affinity column.Antiserum samples are passed through the column and anti-endostatinantibodies remain bound to the column. These antibodies are subsequentlyeluted, collected and evaluated for determination of titer andspecificity.

[0121] The highest titer endostatin antisera is tested to establish thefollowing; a) optimal antiserum dilution for highest specific binding ofthe antigen and lowest non-specific binding, b) the ability to bindincreasing amounts of endostatin peptide in a standard displacementcurve, c) potential cross-reactivity with related peptides and proteins,including endostatin related species, d) ability to detect endostatinpeptides in extracts of plasma, urine, tissues, and in cell culturemedia.

[0122] Kits for measurement of endostatin are also contemplated as partof the present invention. Antisera that possess the highest titer andspecificity and can detect endostatin peptides in extracts of plasma,urine, tissues, and in cell culture media are further examined toestablish easy to use kits for rapid, reliable, sensitive, and specificmeasurement and localization of angiostatin. These assay kits includebut are not limited to the following techniques; competitive andnon-competitive assays, radioimmunoassay, bioluminescence andchemiluminescence assays, fluorometric assays, sandwich assays,immunoradiometric assays, dot blots, enzyme linked assays includingELISA, microtiter plates, antibody coated strips or dipsticks for rapidmonitoring of urine or blood, and immunocytochemistry. For each kit therange, sensitivity, precision, reliability, specificity andreproducibility of the assay are established. Intraassay and interassayvariation is established at 20%, 50% and 80% points on the standardcurves of displacement or activity.

[0123] One example of an assay kit commonly used in research and in theclinic is a radioimmunoassay (RIA) kit. An endostatin RIA is illustratedbelow. After successful radioiodination and purification of endostatinor an endostatin peptide, the antiserum possessing the highest titer isadded at several dilutions to tubes containing a relatively constantamount of radioactivity, such as 10,000 cpm, in a suitable buffersystem. Other tubes contain buffer or preimmune serum to determine thenon-specific binding. After incubation at 4 C. for 24 hours, protein Ais added and the tubes are vortexed, incubated at room temperature for90 minutes, and centrifuged at approximately 2000-2500×g at 4 C. toprecipitate the complexes of antibody bound to labeled antigen. Thesupernatant is removed by aspiration and the radioactivity in thepellets counted in a gamma counter. The antiserum dilution that bindsapproximately 10 to 40% of the labeled peptide after subtraction of thenon-specific binding is further characterized.

[0124] Next, a dilution range (approximately 0.1 pg to 10 ng) of theendostatin peptide used for development of the antiserum is evaluated byadding known amounts of the peptide to tubes containing radiolabeledpeptide and antiserum. After an additional incubation period, forexample, 24 to 48 hours, protein A is added and the tubes centrifuged,supernatant removed and the radioactivity in the pellet counted. Thedisplacement of the binding of radiolabeled endostatin peptide by theunlabeled endostatin peptide (standard) provides a standard curve.Several concentrations of other endostatin peptide fragments,plasminogen, endostatin from different species, and homologous peptidesare added to the assay tubes to characterize the specificity of theendostatin antiserum.

[0125] Extracts of various tissues, including but not limited to,primary and secondary tumors, Lewis lung carcinoma, cultures ofendostatin producing cells, placenta, uterus, and other tissues such asbrain, liver, and intestine, are prepared using extraction techniquesthat have been successfully employed to extract endostatin. Afterlyophilization or Speed Vac of the tisssue extracts, assay buffer isadded and different aliquots are placed into the RIA tubes. Extracts ofknown endostatin producing cells produce displacement curves that areparallel to the standard curve, whereas extracts of tissues that do notproduce endostatin do not displace radiolabeled endostatin from theendostatin antiserum. In addition, extracts of urine, plasma, andcerebrospinal fluid from animals with Lewis lung carcinoma are added tothe assay tubes in increasing amounts. Parallel displacement curvesindicate the utility of the endostatin assay to measure endostatin intissues and body fluids.

[0126] Tissue extracts that contain endostatin are additionallycharacterized by subjecting aliquots to reverse phase HPLC. Eluatefractions are collected, dried in Speed Vac, reconstituted in RIA bufferand analyzed in the endostatin RIA. The maximal amount of endostatinimmunoreactivity is located in the fractions corresponding to theelution position of endostatin.

[0127] The assay kit provides instructions, antiserum, endostatin orendostatin peptide, and possibly radiolabeled endostatin and/or reagentsfor precipitation of bound endostatin—endostatin antibody complexes. Thekit is useful for the measurement of endostatin in biological fluids andtissue extracts of animals and humans with and without tumors.

[0128] Another kit is used for localization of angiostatin in tissuesand cells. This endostatin immunohistochemistry kit providesinstructions, endostatin antiserum, and possibly blocking serum andsecondary antiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This endostatin immunohistochemistry kit permitslocalization of endostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of endostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer.

[0129] This invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

[0130] Identification of an Inhibitor of Capillary Endothelial CellProliferation from Hemangioendothelioma Cells

[0131] A murine hemangioendothelioma cell line, EOMA (Obeso et al.,1990), was evaluated for evidence of the production of inhibitors ofendothelial cell proliferation. Many of the known endogenous inhibitorsof angiogenesis inhibit the in vitro proliferation of endothelial cells.

[0132] Conditioned Media Collection: Cells of the murinehemangioendothelioma cell line EOMA were maintained in DMEM supplementedwith 10% bovine calf serum (BCS) and 1%glutamine-penicillin-streptomycin (GPS) in a 37° C. and 10% CO₂incubator. Conditioned media from EOMA cells (i.e. culture media used togrow EOMA cells) was applied to bovine capillary endothelial cells,stimulated with bFGF, in a 72 hour proliferation assay. The conditionedmedia reversibly inhibited the proliferation of capillary endothelialcells as compared to controls. The pattern of inhibition was consistentwith the presence of inhibitory and stimulatory activity of endothelialcell proliferation (FIG. 1).

EXAMPLE 2

[0133] Inhibitory Activity of Endothelial Cell Proliferation is Not Dueto Angiostatin

[0134] To determine if the inhibitor of capillary endothelial cellproliferation produced by the EOMA cells was angiostatin, pooledconditioned media was applied to a lysine column (lysine conjugated toSepharose™ chromatography beads). Lysine Sepharose binds angiostatin andhas been used for its purification (O'Reilly et al., 1996). Theendothelial cell inhibitory activity was found only in the flow-throughfraction and not in the bound fraction (data not shown). The lack ofbinding of the inhibitory activity to lysine Sepharose suggested thatthe novel inhibitor of endothelial cell proliferation was notangiostatin.

EXAMPLE 3

[0135] Purification of a 20 kDa Protein from the Conditioned Media ofEOMA Cells which Specifically Inhibits Endothelial Cell Proliferation

[0136] Because several angiogenesis inhibitors have an affinity forheparin, we applied the flow-through from the lysine Sepharose column toa heparin Sepharose column. The inhibitory activity bound heparin withrelatively high affinity and was eluted with 0.6-0.8 M NaCl in 10 mMTris pH 7.4, as shown in FIG. 2. To further purify the inhibitoryactivity, the sample was concentrated and applied to a gel filtration(Bio-Rad Bio-Gel P-100 fine gel or Pharmacia Sephacryl S-200HR gel)column (see FIG. 3), followed by several cycles of reverse-phase HPLCwith a C4 column. The inhibitory activity was eluted from the C4 columnwith 40-45% acetonitrile in 0.1% trifluoroacetic acid, as exemplified byFIG. 4. After the final C4 column, the inhibitory activity wasassociated with a protein of molecular mass of approximately 20 kDa(reduced) or 18 kDa (non-reduced), by SDS-PAGE, purified to apparenthomogeneity.

[0137] With respect to Examples 2 and 3, lysine Sepharose, heparinSepharose, Sephacryl S-200 HR gel (Pharmacia, Uppsala, Sweden), Bio-GelP-100 fine polyacrylamide gel (Bio-Rad Laboratories, Richmond, Calif.),and a SynChropak RP-4 (100×4.6 mm) C4 reverse-phase column (Synchrom,Inc., Lafayette, Ind.) were prepared according to the manufacturersrecommendations. A heparin-Sepharose column (50×2.5 cm) was equilibratedwith 50 mM NaCI 10 mM Tris-HCI pH 7.4. Pooled conditioned media wasapplied and the column was washed with the equilibration buffer. Thecolumn was eluted with a continuous gradient of 50 mM-2 M NaCI in 10 mMTris-HCI at pH 7.4 (200 ml total volume) followed by 100 ml of 2 M NaCIin 10 mM Tris-HCI at pH 7.4. Fractions were collected and an aliquot ofeach was applied to capillary endothelial cells. Fractions whichinhibited their proliferation were dialyzed (MWCO=6,000-8,000) againstPBS and concentrated using a 4000 MWCO Nanospin concentrator (GelmanSciences, Ann Arbor, Mich.).

[0138] A Bio-Gel P-100 column or a Sephacryl S-200 HR column (75×1.5 cm)was equilibrated with PBS. The sample from heparin Sepharosechromatography was applied and the column was fluted with theequilibration buffer. Fractions were collected and an aliquot of eachwas applied to endothelial cells. Fractions which inhibited endothelialproliferation were concentrated and dialyzed as above.

[0139] A SynChropak RPG (100×4.6 mm) column was equilibrated withH₂O/0.1% trifluoroacetic acid (TFA). HPLC-grade reagents (Pierce,Rockford, Ill.) were used. The sample from gel filtration chromatographywas applied to the column and the column was fluted with a gradient ofacetonitrile in 0.1% TFA at 0.5 ml/minute and fractions were collected.An aliquot of each was evaporated by vacuum centrifugation, resuspendedin PBS, and applied to capillary endothelial cells. Inhibitory activitywas further purified to apparent homogeneity by at least two subsequentcycles on the SynChropak C4 column.

[0140] To further characterize the 20 kDa inhibitor, we tested it onseveral cell lines of endothelial and non-endothelial origin. For theBCE assay, bovine capillary endothelial cells were obtained and grown aspreviously described (Folkman et al., 1979). For the proliferationassay, cells were washed with PBS and dispersed in a 0.05% solution oftrypsin. A cell suspension (25,000 cells/ml) was made with DMEM+10%BCS+1% GPS, plated onto gelatinized 24-well culture plates (0.5 mewed),and incubated (37° C., 10% CO₂) for 24 hours. The media was replacedwith 0.25 ml of DMEM+5% BCS+1% GPS and the test sample applied. After 20minutes of incubation, media and bFGF were added to obtain a finalvolume of 0.5 ml of DMEM+5% BCS+1% GPS+1 ng/ml bFGF. After 72 hours,cells were dispersed in trypsin, resuspended in Hematall (FisherScientific, Pittsburgh, Pa.), and counted by Coulter counter.

[0141] Non-Endothelial Cell Proliferation Assays

[0142] Bovine aortic smooth muscle (SMC), bovine retinal pigmentepithelial (RPE), mink lung epithelial (MLE), Lewis lung carcinoma(LLC), and EOMA cells and 3T3 fibroblasts were maintained in a 10% CO2and 37° C. incubator. For the proliferation assays, cells were washedwith PBS and were dispersed in a 0.05% solution of trypsin. Optimalconditions for the cell proliferation assays were established for eachdifferent cell type. Fetal calf serum (FCS) was used for the RPE, MLE,and LLC cells and BCS was used for the other cell types. A cellsuspension (20,000 cells/ml for SMC, RPE, MLE; 15,000 cells/ml for 3T3;10,000 cells/ml for LLC, EOMA) was made with DMEM+10% bovine serum+1%GPS, plated onto 24-well culture plates (0.5 ml/well), and incubated(37° C., 10% C02) for 24 hours. The media was replaced with 0.5 ml ofDMEM+5% bovine serum+1% GPS and the test sample applied. After 72 hours,cells were dispersed in trypsin, resuspended in Hematall (FisherScientific, Pittsburgh, Pa.), and counted by Coulter counter.

[0143] Only endothelial cells were significantly inhibited, as shown inTable 2. TABLE 2 EFFECT OF ENDOSTATIN ON ENDOTHELIAL AND NON-ENDOTHELIALCELL PROLIFERATION INHIBITED NON-INHIBITED Bovine capillary Bovineaortic smooth endothelial cells muscle cells Bovine retinal pigmentepithelial cells 3T3 fibroblasts Mink lung epithelial cells EOMAhemangioendothelioma cells Lewis Lung carcinoma cells

[0144] The inhibition was first observed at doses of 100 ng/ml withmaximal inhibition observed at doses of 600 ng/ml or greater. Nosignificant inhibition was seen for cells of non-endothelial origin atdoses 1 log unit higher than those used to inhibit capillary endothelialcell proliferation (data not shown).

EXAMPLE 4

[0145] Microsequence Analysis of the 20 kDa Protein Reveals Identity toa Fragment of Collagen XVIII

[0146] The 20 kDa inhibitor of capillary endothelial cell proliferationfrom the conditioned media was purified to homogeneity, as described inthe above examles, resolved by SDS-PAGE, electroblotted onto PVDF(Bio-Rad, Richmond, Calif.), detected by Ponceau S stain, and excisedfrom the membrane. N-terminal sequence was determined by automated Edmandegradation on an PE/ABD Model 470 A protein sequencer (Foster City,Calif.) operated with gas-phase delivery of trifluoracetic acid.

[0147] Sequence library searches and alignments were performed againstcombined GenBank, Brookhaven Protein, SWISS-PROT, and PIR databases.Searches were performed at the National Center for BiotechnologyInformation through the use of the BLAST network service.

[0148] Microsequence analysis of the inhibitor revealed identity to aC-terminal fragment of collagen XVIII. The molecular cloning andsequence of collagen XVIII was first described by Olsen and hiscoworkers and by Rehn and Pihlajaniemi (Oh et al., 1994; Rehn andPihlajaniemi, 1994). Collagen XVIII is a novel collagen which consistsof an N-terminal region with 3 splice variants (Muragaki et al., 1995;Rehn and Pihlajaniemi, 1995), a series of collagen-like domains withinterruptions, and a 35 kDa C-terminal non-collagenous (NC1) domain. An18-amino acid N-terminal microsequence analysis of the purifiedinhibitor of endothelial cell proliferation confirms that it isidentical to a C-terminal fragment of this NC1 domain (FIG. 5). We havenamed this inhibitory fragment of collagen XVIII “endostatin” and it isincluded in the group of molecules that have endostatin activity.

EXAMPLE 5

[0149] Recombinant Mouse Endostatin (Baculovirus or E. coli) InhibitsEndothelial Cell Proliferation in vitro and Angiogenesis in vivo

[0150] The endothelial proliferation cell inhibitor of the presentinvention can be recombinantly expressed in any system used to expressproteins. Non-limiting examples of such expressions systems includebacterial expression systems, yeast expression systems and insect viralexpression systems.

[0151] Recombinant mouse endostatin was expressed using the BacPAKbaculovirus expression system (CLONTECH Laboratories) following themanufacture's protocol. Briefly, a cDNA fragment encoding the signalsequence and C-terminal part (endostatin region) of mouse collagen XVIIIwas inserted into the pBacPAK8 transfer vector. BacPAK6 viral DNA(expression vector) and plasmid DNA of the pBacPAK8-endostatin clone(modified transfer vector) were then cotransfected into insect Sf21cells and media containing expressed mouse endostatin was collected. TheBacPAK6 was first digested with BSU36 enzyme to make it incompetent forindependent replication. The media containing expressed mouse endostatinwas applied to a 1.5×40 cm heparin Sepharose column which had beenequilibrated with 50 mM NaCl 10 mM Tris pH 7.4. The column was washedwith the equilibration buffer and was then eluted sequentially with 0.2M NaCl, 0.4 M NaCl, 0.6 M NaCl, and 1 M NaCl in 10 mM Tris pH 7.4. Allchromatography was performed at 4° C. The 0.6 M NaCl eluant (whichinhibited bovine capillary endothelial cells in a 72 hour proliferationassay) was dialyzed (6-8000 MWCO) against PBS and then reapplied to theheparin Sepharose column. The column was eluted with a gradient of 50 mMNaCl-1.2 M NaCl in 10 mM Tris pH 7.4. An aliquot of each fraction wasapplied to bovine capillary endothelial cells as above and fractionswhich inhibited proliferation were pooled, dialyzed against PBS, andconcentrated using a Nanospin Plus (Gelman Sciences) centrifugalconcentrator (MWCO=10,000). SDS-PAGE of the concentrated sample revealeda discrete band of apparent M_(r) of 20 kDa.

[0152] Expression and Purification of Recombinant Mouse Endostatin fromE. Coli

[0153] The C-terminal part of the cDNA of collagen XVIII was used toamplify the cDNA of mouse endostatin which was cloned into the pETKH1vector (pET11d derivative) (Studier et al., 1990). Induction resulted inthe production of a fusion protein carrying the amino acid sequenceMARRASVGTD (RRAS=protein kinase A recognition sequence) and 6 histidineresidues at the N-terminus followed by the sequence of mouse endostatin(pTB01#8). The pTB01#8 plasmid was transformed into BL21:DE3 and thefusion protein was purified on Ni⁺²-NTA-beads as described(QiaExpressionist Handbook, Qiagen). Briefly, E. coli were grown untilan O.D.₆₀₀ of 0.8-0.9 and expression of the fusion protein was theninduced for 3 hours with 1 mM IPTG. The bacteria were pelleted andresuspended in 8 M urea, 10 mM Tris-HCl pH 8.0 containing 10 mMimidazole and incubated for 1 hour at room temperature. The suspensionwas centrifuged for 15 minutes at 20,000 g and the supernatant incubatedwith the Ni⁺²-NTA beads for 1 hour at room temperature. The suspensionwas transferred into a column and washed with 8 M urea, 0.1 MNa-phosphate, 10 mM Tris-HCl pH 6.25 containing 10 mM imidazole. Theprotein was eluted with the same buffer containing 250 mM imidazole. Thefractions containing endostatin were extensively dialyzed against PBS.During dialysis, the endostatin precipitated. The precipitatedendostatin was resuspended in PBS, the protein concentration wasadjusted to 2-4 mg/ml, and the endostatin was stored at −20° C. untiluse. For the mouse studies, endostatin was delivered as a suspension inPBS. For the chick chorioallantoic assay, endostatin was furtherdialyzed against water and then lyophilized.

[0154] Recombinant mouse endostatin was produced in both baculovirus andE. coli expression systems. Using sequential heparin Sepharosechromatography, recombinant mouse endostatin was purified to apparenthomogeneity from insect cell media. Ni⁺²-NTA-agarose was used to purifythe E. coli-derived mouse endostatin.

[0155] SDS-PAGE revealed a discrete band of approximately 20 kDa orapproximately 22 kDa (reduced) purified to apparent homogeneity forbaculovirus and E. coli-derived recombinant endostatins, respectively(data not shown). Both were dialyzed against PBS prior to use. Afterdialysis, the material from the E. coli system precipitated and wasdelivered as a suspension for subsequent in vivo studies. Recombinantendostatin from baculovirus specifically inhibited the proliferation ofbovine capillary endothelial cells in a dose-dependent fashion. Theinhibition was seen at doses of 100 ng/ml with maximal inhibitionobserved at doses above 600 ng/ml. No significant inhibition ofproliferation of cells of non-endothelial origin or of the EOMA cellswas observed when endostatin was tested at doses up to 1 log unit higherthan those used to inhibit endothelial cell proliferation.

[0156] The precipitated (un-refolded) material was not testable invitro, because of its insolubility. However, a small percentage wassoluble in PBS during dialysis and this fraction was used for theendothelial cell assays. Furthermore, after refolding, it was solubleand inhibited endothelial proliferation (data not shown). When thissoluble material was applied to endothelial cells, it was found to beinhibitory at concentrations comparable to both the native andbaculovirus-derived endostatin.

[0157] To test for the ability of recombinant mouse endostatin toinhibit in vivo angiogenesis, we used the chick chorioallantoic membrane(CAM) assay (Folkman, 1985; Nguyen et al., 1994 which are incorporatedherein by reference). Briefly, three day old fertilized white Leghorneggs (Spafas, Norwich, Conn.) were cracked, and embryos with intactyolks were placed in 100×20 mm petri dishes (Folkman, 1985). After 3days of incubation (37° C. and 3% CO₂), a methylcellulose (FisherScientific, Fair Lawn, N.J.) disc containing endostatin was applied tothe CAM of individual embryos. The discs were made by desiccation ofendostatin in 10 μl of 0.45% methylcellulose (in H₂O) on teflon rods.After 48 hours of incubation, embryos and CAMs were observed by means ofa stereomicroscope.

[0158] At doses of 10-20 μg/10 μl disc, there was potent inhibition ofin vivo angiogenesis for both the E. coli and the baculovirus-derivedendostatins in all of the tested CAMs (n=5/group). The E. coliderived-endostatin precipitate gradually dissolved over 5 days andproduced a sustained antiangiogenic effect on the implanted CAMs. Incontrast, the soluble baculovirus-derived endostatin dissolved within 24hours and gave a maximal antiangiogenic effect within a period of 48hours. There was no evidence of toxicity in any of the chick embryostested.

[0159] Human Endostatin was produced recombinanty using similar methods.

EXAMPLE 6

[0160] Recombinant Mouse Endostatin Inhibits the Growth of Metastases

[0161] Because tumor growth is angiogenesis dependent, we treated Lewislung carcinoma metastases systematically with recombinant mouseendostatin expressed in the baculovirus system. Animals with Lewis lungcarcinomas of 600-1200 mm³ tumors were sacrificed and the skin overlyingthe tumor was cleaned with betadine and ethanol. In a laminar flow hood,tumor tissue was excised under aseptic conditions. A suspension of tumorcells in 0.9% normal saline was made by passage of viable tumor tissuethrough a sieve and a series of sequentially smaller hypodermic needlesof diameter 22- to 30-gauge. The final concentration was adjusted to1×107 cells/ml and the suspension was placed on ice. After the site wascleaned with ethanol, the subcutaneous dorsa of mice in the proximalmidline were injected with 1×106 cells in 0.1 ml of saline.

[0162] When tumors were 1500 mm³ in size, approximately 14 days afterimplant, the mice underwent surgical removal of the tumor. The incisionwas closed with simple interrupted sutures. From the day of operation,mice received daily intraperitoneal injections of recombinant(baculovirus) mouse endostatin or saline. Mice received 0.3 mg/kg/day ofendostatin once daily via subcutaneous injection. When the control micebecame sick from metastatic disease (i.e., after 13 days of treatment),all mice were sacrificed and autopsied. Lung surface metastases werecounted by means of a stereomicroscope at 4×magnification.

[0163] The growth of Lewis lung carcinoma metastases was almostcompletely suppressed by the systemic administration of endostatin at adose of 0.3 mg/kg/day given subcutaneously (7±3 metastases/mouse, n=4,p<0.001). In contrast, in mice treated with saline after removal of aLewis lung carcinoma primary tumor, lung metastases grew rapidly (77±7metastases/mouse). Lung weight, which reflects tumor burden, was 240±25mg in the endostatin treated mice versus 760±30 mg in the control mice(p<0.001). Further, there was no weight loss or evidence of toxicity inany of the mice treated with endostatin.

EXAMPLE 7

[0164] Recombinant Mouse Endostatin Inhibits the Growth of PrimaryTumors

[0165] The yield of endostatin from the baculovirus system was lowerthan that of the E. coli system, i.e. 1-2 mg/liter versus 30-40mg/liter. We therefore used E. coli-derived endostatin to study theeffect of endostatin therapy on primary tumor growth. We producedrecombinant mouse endostatin from E. coli in sufficient quantity totreat Lewis lung carcinoma primary tumors. The endostatin wasadministered as a suspension of the precipitated purified protein tomice bearing Lewis lung carcinomas of at least 100-200 mm³. The proteinwas purified by conventional means but was not refolded prior to itsadministration to the mice. The injected precipitate was slowly resorbedover 24-48 hours.

[0166] We are unaware of any precedent for the use of an injected depotof non-refolded recombinant protein as a sustained-release method inanimals. Nevertheless, endostatin gradually resorbed in vivo and provedto have potent antiangiogenic activity which resulted in prolongedanti-tumor and antiangiogenic activity. Therefore, these data suggest anovel general method for the controlled release of recombinant proteins.Based on this rationale, we have delivered non-refolded recombinantangiostatin from E. coli with similar success.

[0167] Accordingly, an aspect of the invention is the administration ofrecombinant endostatin or endostatin analogs in an un-refolded state soas to provide a sustained release depot of endothelial cellproliferation inhibiting protein over a period of at least 8 hours,desirably at least 12 hours, more desirably at least 24 hours or atleast 48 hours, depending on the patient and the disease to be treated.Optionally recombinant and un-refolded angiostatin is administered tosimilarly provide a sustained release depot of protein capable ofreleasing angiostatin protein over a period of at least 8 hours,desirably at least 12 hours, more desirably at least 24 hours or atleast 48 hours, depending on the patient and the disease to be treated.

[0168] Mice were implanted with Lewis lung carcinomas as describedabove. Tumors were measured with a dial-caliper and tumor volumes weredetermined using the formula width²×length×0.52, and the ratio oftreated to control tumor volume (T/C) was determined for the last timepoint. After tumor volume was 100-200 mm³ (0.5-1% of body weight), whichoccurred within 3-7 days, mice were randomized into two groups. Onegroup received recombinant mouse endostatin (E. coli) as a suspension inPBS injected subcutaneously at a site distant from the tumor once daily.The other group received comparable injections of the vehicle alone. Theexperiments were terminated and mice were sacrificed and autopsied whenthe control mice began to die.

[0169] The growth of Lewis lung primary tumors was potently suppressedby systemic therapy with endostatin. Increasing the dose of endostatinwas associated with improved efficacy (data not shown). At a dose of 10mg/kg, tumor growth was inhibited by 97% as compared to control micetreated with vehicle alone. At a dose of 20 mg/kg given once daily, intwo separate experiments, there was an almost complete regression ofestablished primary tumors (>99% inhibition, p<0.001). These surprisingand unexpected results are shown in FIGS. 6 and 7.

[0170]FIGS. 8, 9, 10 and 11 demonstrate the effectiveness of recombinantmouse endostatin for inhibiting tumor growth in a variety of differenttumor models. Also demonstrated is the effectiveness of endostatinderived from human for inhibiting tumor growth.

[0171] Immunohistochemical analysis (FIG. 12) of the residual smalltumors showed a potent inhibition of angiogenesis in the endostatintreated tumors. Further, the proliferative index of tumors in theendostatin and saline treated mice was at the same high level in bothgroups while the apoptotic index increased 8-fold after endostatintherapy. Thus, endostatin therapy results in a similar pattern of tumordormancy to the one we have previously described for angiostatin(Holmgren et al., 1995; O'Reilly et al., 1996). Further, there was noevidence of drug-related toxicity in any of the treated mice.

[0172] After discontinuation of endostatin therapy, a tumor recurred atthe primary site within 5-14 days, became vascularized, and eventuallykilled the mice (data not shown). Notably, we found that E. coli-derivedrecombinant mouse endostatin with a C-terminal polyhistidine tag, whichwas expressed. purified and administered in a comparable fashion to theN-terminal tagged product described above did not inhibit angiogenesisin the CAM assay and had no effect on the growth of Lewis lungcarcinomas (data not shown). These data argue strongly that theanti-tumor and antiangiogenic activity of recombinant endostatin are dueto the specific structure of endostatin and not to a contaminant in thesample.

[0173]FIG. 13 shows the results of cycled treatment of Lewis lungcarcinoma with recombinant mouse endostatin derived from E. coli. Theseresults clearly show reproducible endostatin-dependent regression oftumor mass, followed by tumor growth after termination of endostatintreatment.

[0174] These results show that a murine hemangioendothelioma produces anovel and specific 20 kDa inhibitor of endothelial cell proliferation invitro which is also a potent inhibitor of angiogenesis and tumor growthin vivo. The N-terminal sequence of this inhibitor, endostatin, isidentical to a C-terminal fragment of collagen XVIII. Systemicadministration of recombinant endostatin potently inhibits angiogenesis,maintains metastases at a microscopic size, and regresses primary tumorsto less than 1 mm³, a reduction of over 150-fold. For as long as miceare treated there is no regrowth of tumors, no evidence of drugresistance, and no toxicity. It is interesting to note that somefragments of the C-terminal domain of collagen type XVIII that arelonger than endostatin do not inhibit endothelial cell proliferation(data not shown).

[0175] Endostatin was discovered by the same strategy employed to findangiostatin (O'Reilly et al.,1994), i.e., isolation from a tumor. Whileit is counter-intuitive that tumors should be a source of angiogenesisinhibitors, the results reported here seem to validate this approach.

[0176] This leads to the question of why angiogenesis inhibitors shouldbe present in tumors that are angiogenic. One possibility is that aninhibitor could be ‘left-over’ after down-regulation of its productionby a tumor cell undergoing the switch to the angiogenic phenotype. Thisappears to be the case for thrombospondin produced by Li-Fraumeni cellsin which the second allele for p53 is mutated or deleted (Dameron etal., 1994).

[0177] A second possibility is that the proteolytic activity whichaccompanies tumor growth, and which is an important component ofcapillary blood vessel growth, may also mobilize circulatingangiogenesis inhibitors from precursor proteins which are not inhibitorythemselves. Angiostatin for example, inhibits angiogenesis andendothelial cell proliferation while plasminogen does not (O'Reilly etal., 1996; O'Reilly et al., 1994). For endostatin, a similar pattern isrevealed.

[0178] Histology of tumors which regressed under endostatin therapyshowed perivascular cuffing of tumor cells surrounding one or moremicrovessels in which angiogenesis was blocked. Tumor cells displayedhigh proliferation balanced by high apoptosis, with no net gain in tumorsize. These data are consistent with a model of a new type of tumordormancy recently proposed (Holmgren et al., 1995). Furthermore,endostatin inhibited proliferation of endothelial cells in vitro, buthad no effect on Lewis lung carcinoma cells, or other cell typesincluding smooth muscle, epithelium, fibroblasts, and the EOMA cell linefrom which it was purified.

[0179] The fact that a specific inhibitor of endothelial cellproliferation can regress a tumor to a microscopic size and hold it in adormant state, despite the fact that the tumor cells are refractory tothe inhibitor from the outset, indicates that the endothelial populationcan exert powerful growth regulatory control over the tumor cells.

[0180] The results with endostatin support the theory (Folkman, 1996)that for therapeutic purposes, it is fruitful to think about a tumor interms of two distinct cell populations: a tumor cell population and anendothelial cell population, each of which can stimulate growth of theother. Growth of each cell population may be optimally inhibited byagents which selectively or specifically target that cell type, i.e.,cytotoxic chemotherapy and antiangiogenic therapy. Furthermore, combinedtreatment of both cell populations may be better than treatment ofeither cell type alone.

[0181] To test this theory mice seeded with Lewis lung carcinomas, andbearing tumors which had attained a size of approximately 300 mm³, weretreated with a combination therapy comprising angiostatin andendostatin, each at a dose of 20 mg/kg/day for 25 days. Tumors regressedto microscopic levels by about day 10 of treatment. A completelyunexpected finding was that tumors remained regressed and dormant forapproximatley three months, even after all treatment was terminated, asis shown in FIG. 14. Experiments of longer duration indicate that aninitial treatment of tumor with a combination of angiostatin andendostatin causes a very long term dormancy, the actual period of whichis unknown at this time.

[0182] Such long term dormancy is considered a cure to one skilled inthe art. For example, the NIH guideline for deetermining when atreatment is effective as a cancer cure, is that the tumor remaindormant (i.e. not increasing in size) for ten times the normal doublingtime of the tumor. The dormancy length achieved using a combination ofendostatin and angiostatin far exceeds this criteria.

[0183] Accordingly, an important aspect of the invention is acomposition comprising a combination of angiostatin and endostatin, oran endostatin analog, in amounts sufficient to cause long term dormancy,or cure, of angiogenesis-dependent cancers when administered to patientswith angiogenesis-dependent cancers. Administration can be systemically,for example by injection, in which case the dosage is determineddepending upon the patient and the particular cancer, but whichgenerally is at least 0.2 mg/kg/day, desirably at least 2.0 mg/kg/day,more desirably at least 20 mg/kg/day. Generally, the composition isadministered daily for at least 10 days, desirably at least 20 days,more desirably at least 25 days. Alternative systemic administrationroutes include, orally where the composition is formulated, for exampleinto coated microbeads, to protect the protein from inactivatingdigestive environments; transdermally; and via pump.

[0184] Alternatively, different dosages and treatment periods can beused if the composition is administered locally to anangiogenesis-dependent site, such as a tumor. Such administration maybe, for example, surgical implantation or local injection into, or nearby, the site.

EXAMPLE 8

[0185] Isolation of the Putative Receptor for Endostatin

[0186] Both endostatin and angiostatin appear to be specific inhibitorsof endothelial cell proliferation. Therefore, it is likely thatendostatin binds to specific structures exclusively expressed on thesurface of endothelial cells. We are not aware of the existence of anyother specific inhibitors of endothelial cell proliferation.

[0187] Identifying and isolating proteins which specifically bind toendostatin is accompanied by methods well known in the art, for exampleby affinity chromatography and expression cloning.

[0188] Affinity chromatography. Bovine Capillary Endothelial cells (BCE)are radiolabeled with [35S]-methionine, total cell and membrane extractsprepared and applied to affinity columns prepared with endostatin. As anegative control, fibroblast protein extracts are isolated in a similarway. Bound proteins are eluted from the column using a NaCl gradient andthe different fractions are analysed using standard SDS-PAGE andautoradiography. This procedure yields proteins that are tightly boundto the endostatin column and present only in the endothelial cellderived fractions. Comparing the gel electrophoretic patterns of the twocell types reveals expressed proteins unique to the BCE cells. Proteinsequences subsequently are determined and corresponding gene(s) cloned.A cDNA library of bovine capillary endothelial cells, is prepared andscreened with a degenerative oligo based PCR technique to locate thecDNA(s) of the endostatin—specific binding protein(s). Hybridizationusing degenerative oligonucleotides to the corresponding cDNA, is alsoused to identify genes of endostatin binding proteins. Another approachis to raise antibodies against the peptide sequences with methodsdescribed earlier in the Detailed Description and immunoscreen the samelibrary.

[0189] Expression cloning. A cDNA library of BCE cells is prepared.Poly-A mRNA is isolated from BCE cells whose proliferation haspreviously been inhibited by endostatin. These cells express anendostatin binding protein. The corresponding cDNA library istransfected into cells allowing high expression of the various cDNAs.Binding activity of endostatin to cells which express the receptorprotein on the surface is used as a positive selection of these cells.To select for these cells, purified endostatin is labeled with biotinand consequently detected using either streptavidin coupledmagnetic-beads or FACS sorting. Alternatively, an antibody againstendostatin is used for screening. After selection of the positive cells,the corresponding plasmids are isolated, amplified and transfected againinto high expression cells. After several rounds of positive selection,plasmids are analysed for identical inserts using endonuclease digestionand PCR. Using these data, complementation groups are formed, sequencedand analysed with the BLAST network program. In addition to computeranalysis, individual cDNAs are re-transfected into high expression cellsand tested for endostatin binding activity under different conditions(e.g., competition with non-labeled endostatin, time-course of binding,Scatchard analysis, etc. in other words the use of “classical” receptorcharacterization procedures known to those skilled in the art).

EXAMPLE 9

[0190] Determination of the Minimal Region of the Mouse EndostatinProtein Responsible for its Antiangiogenic Activity

[0191] Different PCR primers are designed, the corresponding cDNAscloned into the E. coli expression system, and the different endostatinfragments purified to homogeneity. The full length cDNA is cut from boththe N- and C-terminus. As a first screen the capillary endothelialproliferation assay and the chick embryo assay are used to determine theresidual activity compared to the full length fragment.

EXAMPLE 10

[0192] Determination of the Putative Enzyme(s) which May ReleaseEndostatin from Collagen XVIII

[0193] Collagen XVIII belongs to the non-fibrillar collagen type familyand can be found in three different splicing variants encoding forproteins with 1315-, 1527-, and 1774 amino acid residues (Rehn, PNAS91:4234, 1994). The difference is caused by alterations in theN-terminal part of the gene and therefore all three splicing variantscould potentially be the source of endostatin which itself is a fragmentof the non-collagenous domain 11 (NC11). The function of collagen XVIIIis not known, but because its message is substantially expressed inhighly vascularized organs, a role in perivascular matrix assemblyand/or structure has been proposed (Oh, et al., Genomics, 19:494, 1994).A first clue about the function of collagen XVIII came from thepurification of endostatin as a potent inhibitor of endothelial cellproliferation.

[0194] From this preliminary data and from our initial observation thatendostatin was purified from conditioned medium of ahemangioendothelioma (EOMA), we asked whether the enzyme(s) whichrelease endostatin from collagen XVIII could be identified?

[0195] The last 325 amino acid residues, encoding for the NC11 domain,are expressed in E. coli and the insect cell baculovirus system, thepurified protein is used as a substrate to identify enzymes that clonethis region of collagen XVIII. By PCR, a cDNA fragment encoding the NC11domain is cloned into an E. coli expression vector (pET series) whichallows high expression of the target protein after induction with IPTG.Alternatively, a vector suitable for insect cell expression is used. Theproteins are tagged with the HIS₆-Tag located on the C-terminus forpurification using Ni²⁺-NTA-beads. An Ni²⁺-NTA-alkaline phosphataseconjugate can detect the C-terminus by Western blotting. Anotherconstruct is made which not only has a HIS₆-Tag on the C-terminus, butwill also encode the hemagglutinin (HA-tag on the N-terminus. This isdetected by Western blotting with an HA-specific monoclonal antibody.The N- and C-terminus of the protein followed after incubation with EOMAsupernatant and different metalloproteinase extracts.

[0196] Cleavage product is detected by SDS-PAGE analysis or Westernblotting, the protein is re-purified using the Ni²⁺-NTA beads, elutedwith imidazole, dialyzed against PBS and tested for inhibitor activityin the various in vitro and in vivo assays (e.g., endothelial cellproliferation, chick embryo, and mouse corneal assay). If the purifiedcleavage product shows inhibitory activity, N-terminal amino acidsequencing is performed and compared to the original starting sequenceof endostatin obtained from the EOMA supernatant. Accordingly, thecleavage procedure can be scaled up to purify sufficient protein fortesting in tumor-bearing mice, and to compare this activity to that ofthe full length NC11 domain.

REFERENCES

[0197] The following references are hereby incorporated by referenceherein in their entirety.

[0198] Angiolillo, A. L., Sgadari, C., Taub, D. D., Liao, F., Farber, J.M., Miaheshwari, S., Kleinman, H. K., Reaman, G. H., and Tosato, G.(1995). Human interferon-inducible protein 10 is a potent inhibitor ofangiogenesis in vivo. J. Exp. Med. 182, 155-162.

[0199] Cao, Y., Chen, C., Weatherbee, J. A., Tsang, M., and Folknan, J.(1995). Gro-beta, a C-X-C chemokine, is an angiogenesis inhibitor thatsuppresses the growth of Lewis lung carcinoma in mice. J. Exp. Med. 182,2069-2077.

[0200] Chen, C., Parangi, S., Tolentino, M. J., and Folkman, J. (1995).A strategy to discover circulating angiogenesis inhibitors generated byhuman tumors. Cancer Res. 55, 4230-4233.

[0201] Clapp, C., Martial, J. A., Guzman, R. C., Rentier-Delrue, F., andWeiner, R. l. (1993). The 16-kilodalton N-terminal fragment of humanprolactin is a potent inhibitor of angiogenesis. Endocrinology 133,1292-1299.

[0202] Dameron, K. M., Volpert, O. V., Tainsky, M. A., and Bouck, N.(1994). Control of angiogenesis in fibroblasts by p53 regulation ofthrombospondin-1. Science 265, 1582.

[0203] Folkman, J. (1996). Tumor angiogenesis and tissue factor. NatureMed. 2, 167-168.

[0204] Folkman, J. (1989). What is the evidence that tumors areangiogenesis dependent?. J. Natl. Cancer Inst. 82, 4-6.

[0205] Folkman, J. (1985). Angiogenesis and its inhibitors. In ImportantAdvances in Oncology 1985, V. T. DeVita, S. Hellman, and S. Rosenberg,eds. (Philadelphia: J. B. Lippincott Company), pp. 42-62.

[0206] Folkman, J., Haundenschild, C. C., and Zetter, B. R. (1979).Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci.USA 76, 5217-5221.

[0207] Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992).Identification of programmed cell death in situ via specific labeling ofnuclear DNA fragmentation. J. Cell Biol., 119, 493-501.

[0208] Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M.,Lemons, R. S., Frazier, W.A., and Bouck, N. P. (1990). A tumorsuppressor-dependent inhibitor of angiogenesis is immunologically andfunctionally indistinguishable from a fragment of thrombospondin. Proc.Nat. Acad. Sci. USA. 87, 6624-6628.

[0209] Grant, D. S., Tashiro, K.-l., Sequi-Real, B., Yamada, Y., Martin,G. R., and Kleinman, H. K. (1989). Two different laminin domains mediatethe differentiation of human endothelial cells into capillary-likestructures in vitro. Cell 58, 933-943.

[0210] Gross, J. L., Moscatelli, D., and Rifkin, D. B. (1983). Increasedcapillary endothelial cell protease activity in response to angiogenicstimuli in vitro. Proc. Natl. Acad. Sci. USA 80, 2623-2627.

[0211] Gupta, S. K., Hassel, T., and Singh, J. P. (1995). A potentinhibitor of endothelial cell proliferation is generated by proteolyticcleavage of the chemokine platelet factor 4. Proc. Natl. Acad. Sci. USA92, 7799-7803.

[0212] Holmgren, L., O'Reilly, M. S., and Folkman, J. (1995). Dormancyof micrometastases: balanced proliferation and apoptosis in the presenceof angiogenesis suppression. Nature Med. 1, 149-153.

[0213] Homandberg, G. A., Williams, J. E., Grant, D., B., S., andEisenstein, R. (1985). Heparin-binding fragments of fibronectin arepotent inhibitors of endothelial cell growth. Am. J. Path. 120, 327-332.

[0214] Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y.,Fujita, T., and Kozai, Y. (1991). Suppression of solid tumor growth byimmunoneutralizing monoclonal antibody against human basic fibroblastgrowth factor. Cancer Res. 51, 6180-6184.

[0215] Kandel, J., Bossy-Wetzel, E., Radvany, F., Klagsburn, M.,Folkman, J., and Hanahan, D. (1991). Neovascularization is associatedwith a switch to the export of bFGF in the multistep development offibrosarcoma. Cell 66, 1095-1104.

[0216] Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N.,Phillips, H. S., and Ferrara, N. (1993). Inhibition of vascularendothelial growth factor-induced angiogenesis suppresses tumor growthin vivo. Nature 362, 841-844.

[0217] Maione, T. E., Gray, G. S., Petro, J., Hunt, A. J., Donner, A.L., Bauer, S. I., Carson, H. F., and Sharpe, R. J. (1990). Inhibition ofangiogenesis by recombinant human platelet factor-4 and relatedpeptides. Science 247, 77-79.

[0218] Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., andUllrich, A. (1994). Glioblastoma growth inhibited in vivo by adominant-negative Flk-1 mutant. Nature 367, 576-579.

[0219] Muragaki, Y., Timmons, S., Griffith, C. M., Oh, S. P., Fadel, B.,Quertemmous, T., and Olsen, B.-R. (1995). Mouse col18a1 is expressed ina tissue-specific manner as three alternative variants and is localizedin basement membrane zones. Proc. Natl. Acad. Sci. USA 92, 8763-8767.

[0220] Nelson, J., Allen, W. E., Scott, W. N., Bailie, J. R., Walker,B., and McFerran, N. V. (1995). Murine epidermal growth factor (EGF)fragment (33-42) inhibits both EGF- and laminin-dependent endothelialcell motility and angiogenesis. Cancer Res. 55, 3772-3776.

[0221] Nguyen, M., Shing, Y., and Folkman, J. (1994). Quantitation ofangiogenesis and antiangiogenesis in the chick embryo chorioallantoicmembrane. Microvascular Res. 47, 31-40.

[0222] O'Reilly, M. S., Holmgren, L., Chen, C. C., and Folkman, J.(1996). Angiostatin induces and sustains dormancy of human primarytumors in mice. Nature Med. 2, 689-692.

[0223] O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J.(1994). Angiostatin: A novel angiogenesis inhibitor that mediates thesuppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328.

[0224] Obeso, J., Weber, J., and Auerbach. R. (1990). Ahemangioendothelioma-derived cell line: its use as a model for the studyof endothelial cell biology. Lab. Invest. 63, 259-269.

[0225] Oh, S. K., Kamagata, Y., Muragaki, Y., Timmons, S., Ooshima, A.,and Olsen, B. R. (1994). Isolation and sequencing of cDNAs for proteinswith multiple domains of GlyXaa-Yaa repeats identify a distinct familyof collagenous proteins. Proc. Natl. Acad. Sci. USA 91, 4229-4233.

[0226] Parangi, S., O'Reilly, M., Christofori, G., Holmgren, L.,Grosfeld, J., Folkman, J., and Hanahan, D. (1996). Antiangiogenictherapy of transgenic mice impairs de novo tumor growth. Proc. Natl.Acad. Sci. USA 93, 2002-2007.

[0227] Rastinejad, F., Polverini, P. J., and Bouck, N. P. (1989).Regulation of the activity of a new inhibitor of angiogenesis by acancer suppressor gene. Cell 56, 345-355.

[0228] Rehn, M., and Pihlajaniemi, T. (1994). a1(XVIII), a collagenchain with frequent interruptions in the collagenous sequence, adistinct tissue distribution, and homology with type XV collagen. Proc.Natl. Acad. Sci. USA 91, 4234-4238.

[0229] Rehn, M., and Pihlajaniemi, T. (1995). Identification of threeN-terminal ends of type XVIII collagen chains and tissue-specificdifferences in the expression of the corresponding transcripts. J. Biol.Chem. 270, 4705-4711.

[0230] Sage, E. H., Bassuk, J. A., Vost, J. C., Folkman. M. J., andLane, T. F. (1995). Inhibition of endothelial cell proliferation bySPARC is mediated through a Ca (2+)-binding EF-hand sequence. J. CellBiochem. 57, 127-140.

[0231] Sakamato, N., Iwahana, M., Tanaka, N. G., and Osaka, 8. (1991).Inhibition of angiogenesis and tumor growth by a synthetic lamininpeptide, CDPGYIGSR-NH₂. Cancer Res. 51, 903-906.

[0232] Strieter, R. M., Kunkel, S. L., Arenberg, D. A., Burdick, M. D.,and Polverini, P. J. (1995). Human interferon-inducible protein 10(IP-10), a member of the C-X-C chemokine family, is an inhibitor ofangiogenesis. Biochem. Biophys. Res. Comm. 210, 51-57.

[0233] Studier, W. F., Rosenberg, A. H., Dunn, J. J., and Dudendorf, J.W. (1990). Use of T7 RNA polymerase to direct expression of clonedgenes. Methods Enzymol. 85, 60-89.

[0234] Teicher, B. A., Holden, S. A., Ara, G., Sotomayor, E. A., andDong, H. Z. (1994). Potentiation of cytotoxic cancer therapies byTNP-470 alone and with other antiangiogenic agents. Int. J. Cancer 57,1-6.

[0235] Tolsma, S. S., Volpert, O. V., Good, D. J., Frazier, W. A.,Polverini, P. J., and Bouck, N. (1993). Peptides derived from twoseparate domains of the matrix protein thrombospondin-1 haveantiangiogenic activity. J. Cell Biol.122, 497-511.

[0236] Voest, E. E., Kenyon, B. M., O'Reilly, M. S., Truitt, G.,D'Amato, R. J., and Folkman, J. (1995). Inhibition of angiogenesis invivo by interleukin 12. J. Natl. Cancer Inst. 87, 581-586.

1 2 1 20 PRT Murinae gen. sp. 1 His Thr His Gln Asp Phe Gln Pro Val LeuHis Leu Val Ala Leu Asn 1 5 10 15 Thr Pro Leu Ser 20 2 10 PRT Murinaegen. sp. 2 Met Ala Arg Arg Ala Ser Val Gly Thr Asp 1 5 10

1. Isolated endostatin.
 2. The isolated endostatin of claim 1comprising, an isolated protein that is approximately 18 kDa asdetermined by non-reduced gel electrophoresis, and approximately 20 kDaas determined by reduced gel electrophoresis, wherein the protein can beisolated from the murine hemangioendothelioma EOMA cell line, andwherein the protein is further characterized by its ability tospecifically inhibit proliferating cultured endothelial cells.
 3. Theendostatin of claim 1, wherein the N-terminal amino acid sequence of theprotein has substantial sequence homology to Seq ID No:1.
 4. Theendostatin of claim 1, wherein the protein has substantial seuqencehomology to a C-terminal peptide fragment of collagen type XVIII.
 5. Theendostatin of claim 1 made by a process comprising recombinantlyproducing the protein of claim 1 in a recombinant expression system, andisolating the recombinantly produced protein in its un-refolded form. 6.The endostatin of claim 5, wherein the recombinant expression system isE. coli or baculovirus.
 7. A compound comprising, an isolated nucleicacid sequence coding for endostatin protein.
 8. The compound of claim 7,wherein the endostatin protein is approximately 18 kDa as determined bynon-reduced gel electrophoresis, and approximately 20 kDa as determinedby reduced gel electrophoresis, wherein the protein can be isolated fromthe murine hemangioendothelioma EOMA, and wherein the protein is furthercharacterized by its ability to specifically inhibit proliferatingcultured endothelial cells.
 9. The compound of claim 7, wherein theN-terminal amino acid sequence of the protein has substantial sequencehomology to Seq ID No:1.
 10. The compound of claim 7, wherein theprotein has substantial seuqence homology to a C-terminal peptidefragment of collagen type XVIII.
 11. A compound comprising, an isolatedantibody capable of specifically binding to endostatin protein.
 12. Thecompound of claim 11, wherein the endostatin protein is approximately 18kDa as determined by non-reduced gel electrophoresis, and approximately20 kDa as determined by reduced gel electrophoresis, wherein the proteincan be isolated from the murine hemangioendothelioma EOMA, and whereinthe protein is further characterized by its ability to specificallyinhibit proliferating cultured endothelial cells.
 13. The compound ofclaim 11, wherein the antibody is a monoclonal antibody.
 14. Thecompound of claim 11, wherein the N-terminal amino acid sequence of theendostatin protein has substantial sequence homology to Seq ID No:1. 15.The compound of claim 11, wherein the endostatin protein has substantialsequence homology to a C-terminal peptide fragment of collagen typeXVIII.
 16. An isolated emdostatin made by a process comprising, a.collecting culture media used to grow murine hemangioendothelioma cellline EOMA; and b. fractionating the media by heparin columnchromatography, wherein the isolated endostatin is a protein that isapproximately 18 kDa as determined by non-reduced gel electrophoresis,and approximately 20 kDa as determined by reduced gel electrophores, andthe protein is capable of specifically inhibiting endothelial cellproliferation in cultured cells.
 17. A method of treating anangiogenesis-related disease comprising, administering to a patient inneed of such treatment of the endostatin of claim 1 in an amountsufficient to inhibit angiogenesis.
 18. The method of claim 17, whereinthe endostatin is a recombinantly produced protein, and wherein therecombinantly produced protein is administered in its un-refolded form.19. The method of claim 18, wherein the recombinantly producedendostatin provides a sustained release of the protein over a period ofat least 8 hours.
 20. The method of claim 17, wherein theangiogenesis-related disease is selected from the group consisting ofangiogenesis-dependent cancers; benign tumors; rheumatoid arthritis;psoriasis; ocular angiogenesis diseases; Osler-Webber Syndrome;myocardial angiogenesis; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; wound granulation; intestinaladhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratchdisease and Helobacter pylori ulcers.
 21. The method of claim 20,wherein the angiogenesis-related disease is angiogenesis-dependentcancer.
 22. A method of treating a patient with anangiogenesis-dependent cancer tumor comprising, administering to apatient in need of such treatment of the endostatin of claim 1 in anamount sufficient to cause tumor regression.
 23. The method of claim 22,wherein the endostatin is a recombinantly produced protein, and whereinthe recombinantly produced protein is administered in its un-refoldedform.
 24. The method of claim 23, wherein the recombinantly producedprotein provides a sustained release of the protein for a period of atleast 8 hours.
 25. A method of curing a patient with anangiogenesis-dependent cancer comprising, administering to a patient inneed of such a cure an angiogenesis-dependent cancer curing amount of acomposition comprising, angiostatin combined with the endostatin ofclaim 1, wherein the angiostatin and the endostatin are provided inamounts such that the composition is capable of effectively inhibitingangiogenesis of angiogenesis-dependent cancers when administered topatients with angiogenesis-dependent cancers.
 26. The method of claim25, wherein at least one of angiostatin and endostatin is arecombinantly produced protein, and wherein the recombinantly producedprotein is administered in its un-refolded form.
 27. The method of claim25, wherein the recombinantly produced protein provides a sustainedrelease of the protein for a period of at least 8 hours.
 28. A method ofbirth control comprising, administering to a female an amount of theendostatin of claim 1 sufficient to prevent embryo implantation.
 29. Themethod of claim 28, wherein the endostatin is recombinantly producedprotein, and wherein the recombinantly produced protein is administeredin its un-refolded form.
 30. The method of claim 29, wherein therecombinantly produced protein provides a sustained release of theprotein for a period of at least 8 hours.
 31. A composition comprising,angiostatin combined with the endostatin of claim 1, wherein theangiostatin and the endostatin are provided in amounts such that thecomposition is capable of effectively regressing the tumor mass ofangiogenesis-dependent tumors when administered to patients with anangiogenesis-dependent tumor.
 32. A method of making endostatin proteincomprising, recombinantly expressing a protein that is approximately 18kDa as determined by non-reduced gel electrophoresis, and approximately20 kDa as determined by reduced gel electrophoresis, and which hassubstantial sequence homology to endostatin, the protein being furthercharacterized by its ability to specifically inhibit proliferatingcultured endothelial cells.
 33. The method of claim 32, wherein theendostatin protein is produced in an expression system selected from thegroup consisting of bacterial expression systems, yeast expressionsystems and insect viral expression systems.